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1.
Nature ; 584(7821): 475-478, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32494008

RESUMO

The endoplasmic reticulum (ER) membrane complex (EMC) cooperates with the Sec61 translocon to co-translationally insert a transmembrane helix (TMH) of many multi-pass integral membrane proteins into the ER membrane, and it is also responsible for inserting the TMH of some tail-anchored proteins1-3. How EMC accomplishes this feat has been unclear. Here we report the first, to our knowledge, cryo-electron microscopy structure of the eukaryotic EMC. We found that the Saccharomyces cerevisiae EMC contains eight subunits (Emc1-6, Emc7 and Emc10), has a large lumenal region and a smaller cytosolic region, and has a transmembrane region formed by Emc4, Emc5 and Emc6 plus the transmembrane domains of Emc1 and Emc3. We identified a five-TMH fold centred around Emc3 that resembles the prokaryotic YidC insertase and that delineates a largely hydrophilic client protein pocket. The transmembrane domain of Emc4 tilts away from the main transmembrane region of EMC and is partially mobile. Mutational studies demonstrated that the flexibility of Emc4 and the hydrophilicity of the client pocket are required for EMC function. The EMC structure reveals notable evolutionary conservation with the prokaryotic insertases4,5, suggests that eukaryotic TMH insertion involves a similar mechanism, and provides a framework for detailed understanding of membrane insertion for numerous eukaryotic integral membrane proteins and tail-anchored proteins.


Assuntos
Microscopia Crioeletrônica , Retículo Endoplasmático/enzimologia , Membranas Intracelulares/enzimologia , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Saccharomyces cerevisiae , Sítios de Ligação , Retículo Endoplasmático/química , Retículo Endoplasmático/ultraestrutura , Evolução Molecular , Interações Hidrofóbicas e Hidrofílicas , Membranas Intracelulares/química , Membranas Intracelulares/ultraestrutura , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato
2.
J Med Chem ; 63(8): 4205-4214, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32227946

RESUMO

Influenza and dengue viruses present a growing global threat to public health. Both viruses depend on the host endoplasmic reticulum (ER) glycoprotein folding pathway. In 2014, Sadat et al. reported two siblings with a rare genetic defect in ER α-glucosidase I (ER Glu I) who showed resistance to viral infections, identifying ER Glu I as a key antiviral target. Here, we show that a single dose of UV-4B (the hydrochloride salt form of N-(9'-methoxynonyl)-1-deoxynojirimycin; MON-DNJ) capable of inhibiting Glu I in vivo is sufficient to prevent death in mice infected with lethal viral doses, even when treatment is started as late as 48 h post infection. The first crystal structure of mammalian ER Glu I will constitute the basis for the development of potent and selective inhibitors. Targeting ER Glu I with UV-4B-derived compounds may alter treatment paradigms for acute viral disease through development of a single-dose therapeutic regime.


Assuntos
Dengue/prevenção & controle , Retículo Endoplasmático/efeitos dos fármacos , Inibidores de Glicosídeo Hidrolases/administração & dosagem , Influenza Humana/prevenção & controle , alfa-Glucosidases , Animais , Dengue/tratamento farmacológico , Dengue/enzimologia , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/enzimologia , Relação Dose-Resposta a Droga , Retículo Endoplasmático/enzimologia , Humanos , Influenza Humana/tratamento farmacológico , Influenza Humana/enzimologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Estrutura Secundária de Proteína , alfa-Glucosidases/metabolismo
3.
Proc Natl Acad Sci U S A ; 117(13): 7150-7158, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32170014

RESUMO

Cholesterol biosynthesis is a high-cost process and, therefore, tightly regulated by both transcriptional and posttranslational negative feedback mechanisms in response to the level of cellular cholesterol. Squalene monooxygenase (SM, also known as squalene epoxidase or SQLE) is a rate-limiting enzyme in the cholesterol biosynthetic pathway and catalyzes epoxidation of squalene. The stability of SM is negatively regulated by cholesterol via its N-terminal regulatory domain (SM-N100). In this study, using a SM-luciferase fusion reporter cell line, we performed a chemical genetics screen that identified inhibitors of SM itself as up-regulators of SM. This effect was mediated through the SM-N100 region, competed with cholesterol-accelerated degradation, and required the E3 ubiquitin ligase MARCH6. However, up-regulation was not observed with statins, well-established cholesterol biosynthesis inhibitors, and this pointed to the presence of another mechanism other than reduced cholesterol synthesis. Further analyses revealed that squalene accumulation upon treatment with the SM inhibitor was responsible for the up-regulatory effect. Using photoaffinity labeling, we demonstrated that squalene directly bound to the N100 region, thereby reducing interaction with and ubiquitination by MARCH6. Our findings suggest that SM senses squalene via its N100 domain to increase its metabolic capacity, highlighting squalene as a feedforward factor for the cholesterol biosynthetic pathway.


Assuntos
Esqualeno Mono-Oxigenase/metabolismo , Esqualeno/metabolismo , Regulação Alostérica , Benzilaminas , Colesterol/biossíntese , Retículo Endoplasmático/enzimologia , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Proteostase , Esqualeno Mono-Oxigenase/antagonistas & inibidores , Tiofenos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
4.
Am J Physiol Gastrointest Liver Physiol ; 318(5): G931-G945, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32174134

RESUMO

Helicobacter pylori infection always induces gastritis, which may progress to ulcer disease or cancer. The mechanisms underlying mucosal injury by the bacteria are incompletely understood. Here, we identify a novel pathway for H. pylori-induced gastric injury, the impairment of maturation of the essential transport enzyme and cell adhesion molecule, Na-K-ATPase. Na-K-ATPase comprises α- and ß-subunits that assemble in the endoplasmic reticulum (ER) before trafficking to the plasma membrane. Attachment of H. pylori to gastric epithelial cells increased Na-K-ATPase ubiquitylation, decreased its surface and total levels, and impaired ion balance. H. pylori did not alter degradation of plasmalemma-resident Na-K-ATPase subunits or their mRNA levels. Infection decreased association of α- and ß-subunits with ER chaperone BiP and impaired assembly of α/ß-heterodimers, as was revealed by quantitative mass spectrometry and immunoblotting of immunoprecipitated complexes. The total level of BiP was not altered, and the decrease in interaction with BiP was not observed for other BiP client proteins. The H. pylori-induced decrease in Na-K-ATPase was prevented by BiP overexpression, stopping protein synthesis, or inhibiting proteasomal, but not lysosomal, protein degradation. The results indicate that H. pylori impairs chaperone-assisted maturation of newly made Na-K-ATPase subunits in the ER independently of a generalized ER stress and induces their ubiquitylation and proteasomal degradation. The decrease in Na-K-ATPase levels is also seen in vivo in the stomachs of gerbils and chronically infected children. Further understanding of H. pylori-induced Na-K-ATPase degradation will provide insights for protection against advanced disease.NEW & NOTEWORTHY This work provides evidence that Helicobacter pylori decreases levels of Na-K-ATPase, a vital transport enzyme, in gastric epithelia, both in acutely infected cultured cells and in chronically infected patients and animals. The bacteria interfere with BiP-assisted folding of newly-made Na-K-ATPase subunits in the endoplasmic reticulum, accelerating their ubiquitylation and proteasomal degradation and decreasing efficiency of the assembly of native enzyme. Decreased Na-K-ATPase expression contributes to H. pylori-induced gastric injury.


Assuntos
Retículo Endoplasmático/enzimologia , Células Epiteliais/enzimologia , Mucosa Gástrica/enzimologia , Gastrite/enzimologia , Proteínas de Choque Térmico/metabolismo , Infecções por Helicobacter/enzimologia , Helicobacter pylori/patogenicidade , ATPase Trocadora de Sódio-Potássio/metabolismo , Células Cultivadas , Retículo Endoplasmático/microbiologia , Estabilidade Enzimática , Células Epiteliais/microbiologia , Mucosa Gástrica/microbiologia , Gastrite/genética , Gastrite/microbiologia , Infecções por Helicobacter/genética , Infecções por Helicobacter/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Proteólise , ATPase Trocadora de Sódio-Potássio/genética , Ubiquitinação
5.
Biochem Soc Trans ; 48(1): 199-205, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32065230

RESUMO

Tyrosine kinases are signaling molecules that are common to all metazoans and are involved in the regulation of many cellular processes such as proliferation and survival. While most attention has been devoted to tyrosine kinases signaling at the plasma membrane and the cytosol, very little attention has been dedicated to signaling at endomembranes. In this review, I will discuss recent evidence that we obtained on signaling of tyrosine kinases at the surface of the endoplasmic reticulum (ER), as well as in the lumen of this organelle. I will discuss how tyrosine kinase signaling might regulate ER proteostasis and the implication thereof to general cell physiology.


Assuntos
Retículo Endoplasmático/enzimologia , Proteínas Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Animais , Citosol/enzimologia , Estresse do Retículo Endoplasmático/fisiologia , Humanos , Camundongos , Fosforilação/fisiologia , Proteostase/fisiologia , Resposta a Proteínas não Dobradas
6.
Nature ; 579(7799): 443-447, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32103179

RESUMO

In eukaryotic protein N-glycosylation, a series of glycosyltransferases catalyse the biosynthesis of a dolichylpyrophosphate-linked oligosaccharide before its transfer onto acceptor proteins1. The final seven steps occur in the lumen of the endoplasmic reticulum (ER) and require dolichylphosphate-activated mannose and glucose as donor substrates2. The responsible enzymes-ALG3, ALG9, ALG12, ALG6, ALG8 and ALG10-are glycosyltransferases of the C-superfamily (GT-Cs), which are loosely defined as containing membrane-spanning helices and processing an isoprenoid-linked carbohydrate donor substrate3,4. Here we present the cryo-electron microscopy structure of yeast ALG6 at 3.0 Å resolution, which reveals a previously undescribed transmembrane protein fold. Comparison with reported GT-C structures suggests that GT-C enzymes contain a modular architecture with a conserved module and a variable module, each with distinct functional roles. We used synthetic analogues of dolichylphosphate-linked and dolichylpyrophosphate-linked sugars and enzymatic glycan extension to generate donor and acceptor substrates using purified enzymes of the ALG pathway to recapitulate the activity of ALG6 in vitro. A second cryo-electron microscopy structure of ALG6 bound to an analogue of dolichylphosphate-glucose at 3.9 Å resolution revealed the active site of the enzyme. Functional analysis of ALG6 variants identified a catalytic aspartate residue that probably acts as a general base. This residue is conserved in the GT-C superfamily. Our results define the architecture of ER-luminal GT-C enzymes and provide a structural basis for understanding their catalytic mechanisms.


Assuntos
Microscopia Crioeletrônica , Retículo Endoplasmático/enzimologia , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Biocatálise , Domínio Catalítico , Sequência Conservada , Dolicol Monofosfato Manose/metabolismo , Fosfatos de Dolicol/metabolismo , Glucose/análogos & derivados , Glucose/metabolismo , Glicosiltransferases/deficiência , Técnicas In Vitro , Lipídeos , Proteínas de Membrana/deficiência , Modelos Moleculares , Mutação , Monossacarídeos de Poli-Isoprenil Fosfato/química , Monossacarídeos de Poli-Isoprenil Fosfato/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/genética , Especificidade por Substrato
7.
Adv Exp Med Biol ; 1131: 131-161, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646509

RESUMO

Calcium (Ca2+) is a fundamental regulator of cell fate and intracellular Ca2+ homeostasis is crucial for proper function of the nerve cells. Given the complexity of neurons, a constellation of mechanisms finely tunes the intracellular Ca2+ signaling. We are focusing on the sarco/endoplasmic reticulum (SR/ER) calcium (Ca2+)-ATPase (SERCA) pump, an integral ER protein. SERCA's well established role is to preserve low cytosolic Ca2+ levels ([Ca2+]cyt), by pumping free Ca2+ ions into the ER lumen, utilizing ATP hydrolysis. The SERCA pumps are encoded by three distinct genes, SERCA1-3, resulting in 12 known protein isoforms, with tissue-dependent expression patterns. Despite the well-established structure and function of the SERCA pumps, their role in the central nervous system is not clear yet. Interestingly, SERCA-mediated Ca2+ dyshomeostasis has been associated with neuropathological conditions, such as bipolar disorder, schizophrenia, Parkinson's disease and Alzheimer's disease. We summarize here current evidence suggesting a role for SERCA in the neurobiology of neuropsychiatric and neurodegenerative disorders, thus highlighting the importance of this pump in brain physiology and pathophysiology.


Assuntos
Encéfalo , Retículo Endoplasmático , Doenças do Sistema Nervoso , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Encéfalo/enzimologia , Encéfalo/patologia , Retículo Endoplasmático/enzimologia , Regulação Enzimológica da Expressão Gênica , Homeostase , Humanos , Doenças do Sistema Nervoso/enzimologia , Doenças do Sistema Nervoso/fisiopatologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
8.
Proc Natl Acad Sci U S A ; 117(3): 1533-1542, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31871156

RESUMO

The endoplasmic reticulum (ER) membrane-resident stress sensor inositol-requiring enzyme 1 (IRE1) governs the most evolutionarily conserved branch of the unfolded protein response. Upon sensing an accumulation of unfolded proteins in the ER lumen, IRE1 activates its cytoplasmic kinase and ribonuclease domains to transduce the signal. IRE1 activity correlates with its assembly into large clusters, yet the biophysical characteristics of IRE1 clusters remain poorly characterized. We combined superresolution microscopy, single-particle tracking, fluorescence recovery, and photoconversion to examine IRE1 clustering quantitatively in living human and mouse cells. Our results revealed that: 1) In contrast to qualitative impressions gleaned from microscopic images, IRE1 clusters comprise only a small fraction (∼5%) of the total IRE1 in the cell; 2) IRE1 clusters have complex topologies that display features of higher-order organization; 3) IRE1 clusters contain a diffusionally constrained core, indicating that they are not phase-separated liquid condensates; 4) IRE1 molecules in clusters remain diffusionally accessible to the free pool of IRE1 molecules in the general ER network; 5) when IRE1 clusters disappear at later time points of ER stress as IRE1 signaling attenuates, their constituent molecules are released back into the ER network and not degraded; 6) IRE1 cluster assembly and disassembly are mechanistically distinct; and 7) IRE1 clusters' mobility is nearly independent of cluster size. Taken together, these insights define the clusters as dynamic assemblies with unique properties. The analysis tools developed for this study will be widely applicable to investigations of clustering behaviors in other signaling proteins.


Assuntos
Endorribonucleases/metabolismo , Microscopia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Análise por Conglomerados , Citosol/metabolismo , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Endorribonucleases/química , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/química , Ribonucleases/metabolismo , Transdução de Sinais , Resposta a Proteínas não Dobradas
9.
Adv Exp Med Biol ; 1159: 49-63, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31502199

RESUMO

The majority of enzymes in the sphingolipid (SL) biosynthetic pathway have been identified over the past couple of decades. Despite significant work, and despite their crucial and central roles in SL synthesis, significant information is still lacking concerning the enzymes that catalyze the N-acylation of sphingoid long chain bases, namely the ceramide synthases (CerS), a family of six mammalian genes originally named longevity assurance (Lass) genes. Each of these six endoplasmic reticulum (ER) membrane-bound enzymes utilizes a relatively restricted sub-set of fatty acyl-CoAs for N-acylation, but are far more promiscuous about the use of long chain bases. The reason that mammals and other species have multiple CerS, generating a specific subset of ceramides, is not yet known, but implies an important role for ceramides containing specific fatty acids in cell physiology. In this brief chapter, we will stroll down the CerS lane and discuss what is known, and what is not known, about this important enzyme family.


Assuntos
Ceramidas/biossíntese , Retículo Endoplasmático/enzimologia , Esfingosina N-Aciltransferase/fisiologia , Animais , Ácidos Graxos/química , Esfingolipídeos
10.
Mol Cell ; 76(1): 191-205.e10, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31445887

RESUMO

Normal mitochondrial functions rely on optimized composition of their resident proteins, and proteins mistargeted to mitochondria need to be efficiently removed. Msp1, an AAA-ATPase in the mitochondrial outer membrane (OM), facilitates degradation of tail-anchored (TA) proteins mistargeted to the OM, yet how Msp1 cooperates with other factors to conduct this process was unclear. Here, we show that Msp1 recognizes substrate TA proteins and facilitates their transfer to the endoplasmic reticulum (ER). Doa10 in the ER membrane then ubiquitinates them with Ubc6 and Ubc7. Ubiquitinated substrates are extracted from the ER membrane by another AAA-ATPase in the cytosol, Cdc48, with Ufd1 and Npl4 for proteasomal degradation in the cytosol. Thus, Msp1 functions as an extractase that mediates clearance of mistargeted TA proteins by facilitating their transfer to the ER for protein quality control.


Assuntos
Adenosina Trifosfatases/metabolismo , Retículo Endoplasmático/enzimologia , Mitocôndrias/enzimologia , Membranas Mitocondriais/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Adenosina Trifosfatases/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Proteico , Proteólise , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteína com Valosina/genética , Proteína com Valosina/metabolismo
11.
Genome Biol ; 20(1): 156, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387610

RESUMO

BACKGROUND: Methylation of nucleotides, notably in the forms of 5-methylcytosine (5mC) in DNA and N6-methyladenosine (m6A) in mRNA, carries important information for gene regulation. 5mC has been elucidated to participate in the regulation of fruit ripening, whereas the function of m6A in this process and the interplay between 5mC and m6A remain uncharacterized. RESULTS: Here, we show that mRNA m6A methylation exhibits dynamic changes similar to DNA methylation during tomato fruit ripening. RNA methylome analysis reveals that m6A methylation is a prevalent modification in the mRNA of tomato fruit, and the m6A sites are enriched around the stop codons and within the 3' untranslated regions. In the fruit of the ripening-deficient epimutant Colorless non-ripening (Cnr) which harbors DNA hypermethylation, over 1100 transcripts display increased m6A levels, while only 134 transcripts show decreased m6A enrichment, suggesting a global increase in m6A. The m6A deposition is generally negatively correlated with transcript abundance. Further analysis demonstrates that the overall increase in m6A methylation in Cnr mutant fruit is associated with the decreased expression of RNA demethylase gene SlALKBH2, which is regulated by DNA methylation. Interestingly, SlALKBH2 has the ability to bind the transcript of SlDML2, a DNA demethylase gene required for tomato fruit ripening, and modulates its stability via m6A demethylation. Mutation of SlALKBH2 decreases the abundance of SlDML2 mRNA and delays fruit ripening. CONCLUSIONS: Our study identifies a novel layer of gene regulation for key ripening genes and establishes an essential molecular link between DNA methylation and mRNA m6A methylation during fruit ripening.


Assuntos
Adenosina/análogos & derivados , Regulação da Expressão Gênica de Plantas , Lycopersicon esculentum/genética , RNA Mensageiro/metabolismo , Adenosina/metabolismo , Metilação de DNA , Retículo Endoplasmático/enzimologia , Frutas/genética , Frutas/metabolismo , Regulação Enzimológica da Expressão Gênica , Lycopersicon esculentum/enzimologia , Lycopersicon esculentum/metabolismo , Metilação , Mutação , Motivos de Nucleotídeos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estabilidade de RNA , RNA Mensageiro/química
12.
PLoS Genet ; 15(6): e1008196, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31173582

RESUMO

Covalent intermolecular cross-linking of collagen is essential for tissue stability. Recent studies have demonstrated that cyclophilin B (CypB), an endoplasmic reticulum (ER)-resident peptidyl-prolyl cis-trans isomerase, modulates lysine (Lys) hydroxylation of type I collagen impacting cross-linking chemistry. However, the extent of modulation, the molecular mechanism and the functional outcome in tissues are not well understood. Here, we report that, in CypB null (KO) mouse skin, two unusual collagen cross-links lacking Lys hydroxylation are formed while neither was detected in wild type (WT) or heterozygous (Het) mice. Mass spectrometric analysis of type I collagen showed that none of the telopeptidyl Lys was hydroxylated in KO or WT/Het mice. Hydroxylation of the helical cross-linking Lys residues was almost complete in WT/Het but was markedly diminished in KO. Lys hydroxylation at other sites was also lower in KO but to a lesser extent. A key glycosylation site, α1(I) Lys-87, was underglycosylated while other sites were mostly overglycosylated in KO. Despite these findings, lysyl hydroxylases and glycosyltransferase 25 domain 1 levels were significantly higher in KO than WT/Het. However, the components of ER chaperone complex that positively or negatively regulates lysyl hydroxylase activities were severely reduced or slightly increased, respectively, in KO. The atomic force microscopy-based nanoindentation modulus were significantly lower in KO skin than WT. These data demonstrate that CypB deficiency profoundly affects Lys post-translational modifications of collagen likely by modulating LH chaperone complexes. Together, our study underscores the critical role of CypB in Lys modifications of collagen, cross-linking and mechanical properties of skin.


Assuntos
Ciclofilinas/química , Lisina/química , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/química , Pele/enzimologia , Animais , Colágeno Tipo I/biossíntese , Colágeno Tipo I/genética , Ciclofilinas/genética , Ciclofilinas/ultraestrutura , Retículo Endoplasmático/química , Retículo Endoplasmático/enzimologia , Glicosilação , Heterozigoto , Hidroxilação , Lisina/genética , Espectrometria de Massas , Camundongos , Camundongos Knockout , Microscopia de Força Atômica , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Processamento de Proteína Pós-Traducional/genética , Pele/química
13.
New Phytol ; 223(4): 1904-1917, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31087404

RESUMO

Choline kinase catalyzes the initial reaction step of choline metabolism that produces phosphocholine, a prerequisite for the biosynthesis of a primary phospholipid phosphatidylcholine. However, the primary choline kinase and its role in plant growth remained elusive in seed plants. Here, we showed that Arabidopsis CHOLINE/ETHANOLAMINE KINASE 1 (CEK1) encodes functional CEK that prefers choline than ethanolamine as a substrate in vitro and affects contents of choline and phosphocholine but not phosphatidylcholine in vivo. CEK1 is localized at endoplasmic reticulum (ER); upon tunicamycin-induced ER stress, a null mutant of CEK1 showed hypersensitive phenotype in seedlings, albeit with no enhanced choline kinase activity. Our results demonstrate that CEK1 is a primary ER-localized choline kinase in vivo that is required for ER stress tolerance possibly through the modulation of choline metabolites.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/enzimologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Colina/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Marcadores Genéticos , Análise do Fluxo Metabólico , Mutação/genética , Especificidade de Órgãos/efeitos dos fármacos , Fenótipo , Plântula/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos , Tunicamicina/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos
14.
Glycobiology ; 29(7): 530-542, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-30976784

RESUMO

The endoplasmic reticulum (ER) contains both α-glucosidases and α-mannosidases which process the N-linked oligosaccharides of newly synthesized glycoproteins and thereby facilitate polypeptide folding and glycoprotein quality control. By acting as structural mimetics, iminosugars can selectively inhibit these ER localized α-glycosidases, preventing N-glycan trimming and providing a molecular basis for their therapeutic applications. In this study, we investigate the effects of a panel of nine iminosugars on the actions of ER luminal α-glucosidase I and α-glucosidase II. Using ER microsomes to recapitulate authentic protein N-glycosylation and oligosaccharide processing, we identify five iminosugars that selectively inhibit N-glycan trimming. Comparison of their inhibitory activities in ER microsomes against their effects on purified ER α-glucosidase II, suggests that 3,7a-diepi-alexine acts as a selective inhibitor of ER α-glucosidase I. The other active iminosugars all inhibit α-glucosidase II and, having identified 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) as the most effective of these compounds, we use in silico modeling to understand the molecular basis for this enhanced activity. Taken together, our work identifies the C-3 substituted pyrrolizidines casuarine and 3,7a-diepi-alexine as promising "second-generation" iminosugar inhibitors.


Assuntos
Arabinose/farmacologia , Retículo Endoplasmático/enzimologia , Inibidores de Glicosídeo Hidrolases/farmacologia , Imino Furanoses/farmacologia , Alcaloides de Pirrolizidina/farmacologia , Álcoois Açúcares/farmacologia , alfa-Glucosidases/metabolismo , Animais , Arabinose/química , Cães , Inibidores de Glicosídeo Hidrolases/química , Humanos , Imino Furanoses/química , Camundongos , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Alcaloides de Pirrolizidina/química , Álcoois Açúcares/química
15.
Elife ; 82019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30973820

RESUMO

Unfolded protein responses (UPRs) safeguard cellular function during proteotoxic stress and aging. In a previous paper (Lehrbach and Ruvkun, 2016) we showed that the ER-associated SKN-1A/Nrf1 transcription factor activates proteasome subunit expression in response to proteasome dysfunction, but it was not established whether SKN-1A/Nrf1 adjusts proteasome capacity in response to other proteotoxic insults. Here, we reveal that misfolded endogenous proteins and the human amyloid beta peptide trigger activation of proteasome subunit expression by SKN-1A/Nrf1. SKN-1A activation is protective against age-dependent defects caused by accumulation of misfolded and aggregation-prone proteins. In a C. elegans Alzheimer's disease model, SKN-1A/Nrf1 slows accumulation of the amyloid beta peptide and delays adult-onset cellular dysfunction. Our results indicate that SKN-1A surveys cellular protein folding and adjusts proteasome capacity to meet the demands of protein quality control pathways, revealing a new arm of the cytosolic UPR. This regulatory axis is critical for healthy aging and may be a target for therapeutic modulation of human aging and age-related disease.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Ligação a DNA/metabolismo , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/metabolismo , Longevidade , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas , Doença de Alzheimer/patologia , Animais , Modelos Animais de Doenças , Humanos
16.
Am J Physiol Heart Circ Physiol ; 316(6): H1323-H1331, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30901276

RESUMO

The type 2a sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA2a) plays a key role in Ca2+ regulation in the heart. However, available techniques to study SERCA function are either cell destructive or lack sensitivity. The goal of this study was to develop an approach to selectively measure SERCA2a function in the cellular environment. The genetically encoded Ca2+ sensor R-CEPIA1er was used to measure the concentration of Ca2+ in the lumen of the endoplasmic reticulum (ER) ([Ca2+]ER) in HEK293 cells expressing human SERCA2a. Coexpression of the ER Ca2+ release channel ryanodine receptor (RyR2) created a Ca2+ release/reuptake system that mimicked aspects of cardiac myocyte Ca2+ handling. SERCA2a function was quantified from the rate of [Ca2+]ER refilling after ER Ca2+ depletion; then, ER Ca2+ leak was measured after SERCA inhibition. ER Ca2+ uptake and leak were analyzed as a function of [Ca2+]ER to determine maximum ER Ca2+ uptake rate and maximum ER Ca2+ load. The sensitivity of this assay was validated by analyzing effects of SERCA inhibitors, [ATP]/[ADP], oxidative stress, phospholamban, and a loss-of-function SERCA2a mutation. In addition, the feasibility of using R-CEPIA1er to study SERCA2a in a native system was evaluated by using in vivo gene delivery to express R-CEPIA1er in mouse hearts. After ventricular myocyte isolation, the same methodology used in HEK293 cells was applied to study endogenous SERCA2a. In conclusion, this new approach can be used as a sensitive screening tool to study the effect of different drugs, posttranslational modifications, and mutations on SERCA function. NEW & NOTEWORTHY The aim of this study was to develop a sensitive approach to selectively measure sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) function in the cellular environment. The newly developed Ca2+ sensor R-CEPIA1er was used to successfully analyze Ca2+ uptake mediated by recombinant and native cardiac SERCA. These results demonstrate that this new approach can be used as a powerful tool to study new mechanisms of Ca2+ pump regulation.


Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/enzimologia , Miócitos Cardíacos/enzimologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/enzimologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Transporte Biológico , Técnicas Biossensoriais , Proteínas de Ligação ao Cálcio/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Mutação , Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Retículo Sarcoplasmático/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Fatores de Tempo
17.
Elife ; 82019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30865586

RESUMO

Misfolded proteins in the endoplasmic reticulum (ER) activate the unfolded protein response (UPR), which enhances protein folding to restore homeostasis. Additional pathways respond to ER stress, but how they help counteract protein misfolding is incompletely understood. Here, we develop a titratable system for the induction of ER stress in yeast to enable a genetic screen for factors that augment stress resistance independently of the UPR. We identify the proteasome biogenesis regulator Rpn4 and show that it cooperates with the UPR. Rpn4 abundance increases during ER stress, first by a post-transcriptional, then by a transcriptional mechanism. Induction of RPN4 transcription is triggered by cytosolic mislocalization of secretory proteins, is mediated by multiple signaling pathways and accelerates clearance of misfolded proteins from the cytosol. Thus, Rpn4 and the UPR are complementary elements of a modular cross-compartment response to ER stress.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas , Retículo Endoplasmático/enzimologia , Biogênese de Organelas
18.
Mol Cell ; 74(1): 45-58.e7, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30846317

RESUMO

Cells require a constant supply of fatty acids to survive and proliferate. Fatty acids incorporate into membrane and storage glycerolipids through a series of endoplasmic reticulum (ER) enzymes, but how these enzymes are regulated is not well understood. Here, using a combination of CRISPR-based genetic screens and unbiased lipidomics, we identified calcineurin B homologous protein 1 (CHP1) as a major regulator of ER glycerolipid synthesis. Loss of CHP1 severely reduces fatty acid incorporation and storage in mammalian cells and invertebrates. Mechanistically, CHP1 binds and activates GPAT4, which catalyzes the initial rate-limiting step in glycerolipid synthesis. GPAT4 activity requires CHP1 to be N-myristoylated, forming a key molecular interface between the two proteins. Interestingly, upon CHP1 loss, the peroxisomal enzyme, GNPAT, partially compensates for the loss of ER lipid synthesis, enabling cell proliferation. Thus, our work identifies a conserved regulator of glycerolipid metabolism and reveals plasticity in lipid synthesis of proliferating cells.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Retículo Endoplasmático/enzimologia , Glicerídeos/biossíntese , Glicerol-3-Fosfato O-Aciltransferase/metabolismo , Lipogênese , Células 3T3 , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proliferação de Células , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/patologia , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica , Glicerol-3-Fosfato O-Aciltransferase/genética , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Células Jurkat , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Camundongos , Ácido Palmítico/toxicidade , Ligação Proteica
19.
PLoS One ; 14(3): e0214118, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30889231

RESUMO

The enzymes GALNTs add GalNAc sugar to Ser and Thr residues, forming the Tn glycan. GALNTs are activated by trafficking from Golgi to ER, a process driven by the Src kinase and negatively regulated by ERK8. This GALNTs activation (aka GALA) pathway induces high Tn levels and is a key driver of liver tumor growth. Recently, Tabak and colleagues have contested our previous data that EGF stimulation can induce GALNTs relocation. Here, we show that relocation induced by EGF is actually detectable in the very images acquired by Tabak et al. Furthermore, we show that over-expression of EGFR strongly enhances EGF-induced relocation and that EGFR appears required to drive relocation induced by ERK8 depletion. Direct co-localisation of GALNT with the ER marker Calnexin is observed after EGF stimulation. We furthermore propose that quantification of O-glycosylation of the ER resident protein PDIA4 provides a mean to quantify GALA independently of imaging. In sum, we demonstrate that the claimed non-reproducibility was due to experimental imaging conditions, that EGFR is indeed a driver of GALA and propose additional markers to facilitate the study of this pathway.


Assuntos
Retículo Endoplasmático/enzimologia , N-Acetilgalactosaminiltransferases/metabolismo , Retículo Endoplasmático/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glicosilação , Células HEK293 , Células HeLa , Humanos , N-Acetilgalactosaminiltransferases/genética , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo
20.
Mol Biol Cell ; 30(9): 1069-1084, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30785834

RESUMO

P5A ATPases are expressed in the endoplasmic reticulum (ER) of all eukaryotic cells, and their disruption results in severe ER stress. However, the function of these ubiquitous membrane proteins, which belong to the P-type ATPase superfamily, is unknown. We purified a functional tagged version of the Saccharomyces cerevisiae P5A ATPase Spf1p and observed that the ATP hydrolytic activity of the protein is stimulated by phosphatidylinositol 4-phosphate (PI4P). Furthermore, SPF1 exhibited negative genetic interactions with SAC1, encoding a PI4P phosphatase, and with OSH1 to OSH6, encoding Osh proteins, which, when energized by a PI4P gradient, drive export of sterols and lipids from the ER. Deletion of SPF1 resulted in increased sensitivity to inhibitors of sterol production, a marked change in the ergosterol/lanosterol ratio, accumulation of sterols in the plasma membrane, and cytosolic accumulation of lipid bodies. We propose that Spf1p maintains cellular sterol homeostasis by influencing the PI4P-induced and Osh-mediated export of sterols from the ER.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Esteróis/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/metabolismo , Homeostase , ATPases do Tipo-P/metabolismo , Filogenia , Receptores de Esteroides/metabolismo , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos
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